Field of the Invention
The field of the present invention relates to a method, an apparatus and a computer program product for the imaging of a labeled biological sample.
Brief Description of the Related Art
A number of so-called “super-resolution” imaging technologies have recently emerged. These super-resolution imaging technologies include stimulated emission depletion (STED), photo-activated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM). These super-resolution imaging technologies enable the imaging of labeled biological samples, by separating the emission of fluorophore labels attached to the biological samples in space or time, thus overcoming the resolution limit of conventional light microscopes. Methods like PALM and STORM activate only a few of the fluorophore labels, followed by fitting. These methods assume that the emission (fluorescence) comes from a single point. STED achieves this by turning of neighboring ones of the emitters (fluorophores) by using a second, donut-shaped STED laser. Such prior art methods therefore localize only single labeled molecules or use a spatially non-uniform illumination and a non-linear photo-response. Given the impact on biological research, the super-resolution imaging technology has thus gained great attention. Assembly of a super-resolution device and running the super-resolution device or acquiring an integrated solution can be expensive and customized systems have been shown to currently outperform commercial ones.
Confocal laser scanning microscopy is one example of a method for obtaining high resolution optical images of a complex object with a degree of depth selectivity. The complex object can be, but is not limited to, a biological specimen. The optical images of the complex object are acquired by a photo-detection device and are subsequently reconstructed by a computer. This computer reconstruction allows a generation of a 3-D reconstruction of the complex object.
In a confocal laser scanning microscope, a laser light beam passes through an aperture and the laser light beam is subsequently focused by an objective lens into a small volume within or onto a surface of the biological specimen. The biological specimen may be labeled with a fluorophore such as fluorescent molecules, antibodies or fluorescent particles. Labeling of the biological specimen with the fluorophore is particularly common for biological applications of microscopy. Scattered and reflected laser light as well as any fluorescent light from the fluorophore of the labeling of the biological specimen is collected by the objective lens. Consequently a beam splitter separates the collected light based on its spectral characteristic. The collected light is than detected by a sensor, such as a light sensitive device. This light sensitive device used in confocal microscopy is typically a point detector such as a photomultiplier. In a spinning disk microscope the light sensitive device can be a charge coupled device (CCD). Moreover in front of the light sensitive device there may be a filter to allow selective passage of light with a specific fluorescent wavelength whilst blocking the laser light beam of the original wave length (i.e. the scattered and reflected laser light). The light sensitive device transforms the light signal into an electrical signal that is passed to a computer where the electrical signal is stored in an image memory in the computer.
Several examples of imaging systems are known in the art in which a time series of images are made of a particular object and difference images are generated. For example, U.S. Pat. No. 4,542,459 teaches a matched filter for x-ray hybrid subtraction in which low energy and high energy x-ray exposures are made before and after an injected x-ray contrast medium arrives at a blood vessel of interest. The '459 patent teaches two different procedures for obtaining a sequence of x-ray images that yield signals or data representative of a hybrid subtraction image. The method of '459 can be used to eliminate signals due to bone and ensure that signals representative of the blood vessel filled with the x-ray contrast medium remain.
U.S. Pat. No. 6,061,476 teaches a method to detect an object in which two images are taken of a particular object at a different time and one image is subtracted from the other image. The resulting subtracted image is separated into two components: a positive difference image and a negative difference image. The two components are processed to determine whether—the object is present or not. In the example given in the '476 patent, the method is used to inspect solder paste as to whether the object mayor may not be present.